The present invention relates to a method for accurately detecting fluorophore-labeled biological substances, especially, to a method for preventing erroneous results due to unexpected dusts or impurities, for example, and to a method for accurately measuring the fluorescence of a sample solution when DNA is amplified, for example, and an apparatus thereof.
Analytical techniques for DNA, protein, and the like are important in the fields of medicine and biology including gene analysis and gene diagnosis. Especially in recent years, methods and apparatuses for inspection and analysis using DNA microarrays (also referred to as DNA chips, for example), protein chips, or the like have attracted attention. The microarrays employ substrates comprising glass, for example. They are divided into a plurality (several hundreds to tens of millions) of fields, and probes for target (usually, different types of) DNA, for example, are immobilized on each field, thereby preparing each field as a minute reaction field. By causing them to react with a specimen, the target DNA in the specimen, protein, or the like is bonded to the aforementioned immobilized probes and is then captured, thereby conducting quantification. Usually, a microscope (confocal fluorescence microscope)-like apparatus referred to as a scanner is used to read fluorescence intensity emitted by the fluorescent label of the target DNA captured in each reaction field of the DNA microarrays (see Patent Document 2, for example). The apparatus irradiates an excitation light, such as a laser, onto the arrays, separates generated fluorescence from the excitation light using a dispersion element such as an interference filter, and detects fluorescence intensity via a light detector, thereby conducting quantitative and qualitative comparisons regarding captured target DNA, protein, or the like.
Regarding the detection, quantitative determination, or the like of a target nucleotide of DNA, for example, real-time fluorescence detection during amplification using a microplate, a microtube, or the like, other than the microarray is also known (see Patent Document 4).
In fluorescence measurement using the microarray, the adhesion of dust poses a great problem. Since the fluorescence intensity of a portion where dust is attached does not show an accurate value, it must be excluded from a measurement value. Usually, spots of the microarray have a size of about 100-micron diameter, and a piece of dust has a size of not more than several microns in many cases. Thus, even when a single piece of dust exists in a certain spot, by removing a signal of the portion alone, the fluorescence measurement of the spot becomes possible in accordance with the fluorescence intensity of the rest field. The spot density of microarrays has been gradually increased and the diameter of spots has become smaller. However, as the spot diameter has become smaller, the size of the spot diameter has no difference with that of dust, so that measurement via the aforementioned method becomes difficult. The same applies to a flaw in a substrate.
Patent Document 2 discloses a method for measuring the properties of a sample using fluorescence from a sample along with a reflected light of an excitation light.
A method for detecting fluorescence during amplification in a container is also performed in general. Usually, a microplate, a microtube, or the like is used as a reaction container and a detection container. Also, Patent Document 1 discloses an extraction-detection module (hereafter referred to as a centrifugal module) comprising a rotatable cartridge-like structure provided with a capturing portion for capturing DNA, virus RNA, or the like in a sample solution such as blood, and with a reagent holding portion for separately holding various types of reagents, for example. The centrifugal module feeds a sample solution via centrifugal force generated through rotation. The present module is an effective device by which it is capable of, once a sample such as blood is set, conducting all reactions and detection in the module, and the possibility of contamination to the outside is substantially low.
However, neither of the aforementioned documents discloses judgment concerning whether fluorescence from a sample is measured in a purely normal state.    Patent Document 1: WO 03/059484    Patent Document 2: JP Patent Publication (Kokai) No. 2002-310886 A    Patent Document 3: JP Patent Publication (Kokai) No. 2002-181708 A    Patent Document 4: JP Patent Publication (Kokai) No. 2002-189860 A